Our work is focussed on determining the pathway and understanding the mechanism of protein folding. We are engaged in two main projects. The first is determination of the kinetic pathway of folding of ribonuclease A. There are at least 3 forms of the unfolded protein, caused perhaps by the cis-trans isomerization of proline residues after unfolding, and we study the refolding pathway of the major slow-folding form. In previous work we have shown that structural intermediates are well-populated transiently during folding at 0 degree 10 degrees C, and that numerous peptide NH protons are protected against exchange with H2O before the tertiary structure is formed. By a D20 trapping method, followed by 2D 1H-NMR after folding is complete, we are determining the structural locations of the protected peptide NH protons. In future work we will use pulse exchange experiments to measure the degree of protection of each protected proton as a function of time during folding. The second main project is to find out if apomyoglobin is a suitable system for testing the framework model of folding and to investigate the role of the helix:helix pairing sites during folding. Privalov has found a prominent equilibrium intermediate in the folding of sperm whale apomyoglobin, whose properties resemble the so-called "molten globule state". We find that human apomyoglobin does not show this intermediate although dog and horse myoglobin do show it, and we propose to use site-directed mutagenesis to find out which amino acid residues are responsible. We will also use the "medium-resolution method" of Rosa and Richards (1979) to find out which alpha-helices are present as judged by protected peptide NH protons.